Method of producing regioselectively substituted esters of oligo- and polysaccharides

ABSTRACT

A regioselectively substituted member selected from the group consisting of oligo-saccharide ester and polysaccharide ester is disclosed. The ester has a partial average degree of substitution AS at the C2 position of its anhydroglucose unit of at least 90% relative to the total AS. Also disclosed is a method of producing the regioselectively substituted ester. The method entails reacting in the presence of a catalyst a dissolved or a highly swollen oligo-saccharide or polysaccharide with an esterification reagent.

[0001] This invention relates to esters of oligo- and polysaccharideswhich are regioselectively substituted, predominantly at the C2 positionof the anhydroglucose unit (AGU), and also relates to a method ofproducing them using enzymes and/or defined salts as catalysts for esterformation. Regioselectively substituted esters of this type have basicproperties which are different from those of conventional, randomlyfunctionalised products.

[0002] It is known that when oligo- and polysaccharides are subjected toesterification by conventional methods, products are formed whichexhibit a random distribution of the esters groups within the AGU andalong the chain. This randomness depends on the accessibility toelectrons or on the spatial accessibility of the individual hydroxylgroups. Thus in starch, for example, the hydroxyl group in the C6position is a primary group which is very sterically accessible andwhich thus exhibits the highest accessibility during heterogeneousreactions in particular. The hydroxyl function of the C2 position formsthe basis of the electronic effect of the adjacent glycoside bond and ofthe electron-attracting properties of the ring oxygen. In homogeneousprocesses, it has been shown that the C2 position reacts first for thisreason. In none of the aforementioned situations, however, is thecomplete reaction of only one hydroxyl group achieved.

[0003] By utilising differences in accessibilities such as these,hydroxyl groups can be selectively blocked with the aid of protectivegroups, which are generally bulky and can easily be split off, so thatregioselective derivatives are formed in subsequent reactions.

[0004] Examples of protective groups such as these includetriphenylmethyl groups or bulky organosilicon entities such as t-hexyl-or tert-butyl-dimethylsilyl groups.

[0005] However, this type of synthesis has the decisive disadvantagethat at least two additional reaction steps are necessary due to theintroduction of the protective group and the separation thereof. Otherdisadvantages are the fact that the separation of the protective groupsis sometimes incomplete, which means that the cleavage products whichare formed thereby, and which are sometimes toxic, have to be removedwithout leaving a residue, as well as the breakdown of thepolysaccharide chain which is possible under the conditions of cleavageand which changes the properties of the product.

[0006] When a plurality of reactive centres, e.g. hydroxyl groups,exists in a molecule, enzymes are capable of catalysing direct,selective esterification reactions. In this connection, each enzyme hasa certain folded (native) structure which is essential for its specificbiocatalytic activity in the physiological medium concerned. It has beenshown in numerous publications, however, that many enzymes are alsoactive in organic solvents, i.e. are active irrespective of their nativestructure, size and function. Enzymes generally exhibit a high activityin nonpolar solvents, whereas only very low activities are found inrelatively polar media (Biotechnol. Bioeng. 30 (1987), 81-87). Enzymesare insoluble in the latter organic solvents.

[0007] Numerous enzyme-catalysed reactions of this type have alreadybeen carried out on low molecular weight mono- and disaccharides inorganic solvents (FEMS Microbiol. Rev. 16 (1995), 193-211; J. Prakt.Chem. 335 (1993), 109-127 Synthesis 1992, 895-910; WO 97/36000; WO95/23871). In these reactions lipases have primarily been used as theenzymes, although esterases and proteases have also been used, andsolvents such as tetrahydrofuran, pyridine and N,N-dimethylformamidehave been employed. Enzymatic esterifications can also be effected in anaqueous buffer solution. One disadvantage here is that the acylatingreagent, which has a character similar to that of a fatty acid, isinsoluble in the aqueous buffer solution and can thus only be suspendedtherein. The substrate which is to be esterified is present in dissolvedform (DE-A-24 30 944, 1992; JP-A-63191802).

[0008] Glucans generally result in the formation of esters at primaryhydroxyl groups. In some cases, esterification also occurs at secondaryhydroxyl groups (J. am. Chem. Soc. 109 (1987), 3977-3981; Enzyme Microb.Technol. 20 (1997), 225-228). Compounds which compriseelectron-attracting groups are generally used for the aforementionedenzymatic esterifications, such as vinyl esters (Biotechnol. Lett. 19(1997), 511-514) or trihalogenoethyl esters (Tetrahedron 54 (1998),3971-3982); esterification by a vinyl ester constitutes an irreversiblereaction, since the vinyl alcohol which is formed is removed asacetaldehyde from the reaction equilibrium. Other reactive compoundsinclude carboxylic anhydrides and esters of carbonic acid.

[0009] Diesters of dicarboxylic acids have also been used for reactionwith mono- and disaccharides. In this manner, it has proved possible tosynthesise new saccharide-based copolymers, as disclosed in U.S. Pat.Nos. 5,270,421 and 5,618,933.

[0010] In the publications described above, the substrate and theacylating reagent are generally dissolved in an organic solvent, inwhich the enzyme is then suspended. Enzymatic esterifications cannot beeffected on polysaccharides, particularly glucans, by the methods citedabove, since the undissolved or unswollen polymer is not accessible tothe enzyme or to enzyme catalysis. Polysaccharides can be esterifiedenzymatically in a heterogeneous phase, however (WO 96/13632, DE-A-34 30944). These heterogeneous reactions only proceed at the surface of thepolymer particles, due to which inhomogeneously esterifed polysaccharidederivatives are formed, i.e. polysaccharide derivatives which comprise anon-uniform distribution of substituents along the polymer chain. Otherdisadvantages of this method of esterification are that low yields ofproduct are obtained and products are formed which comprise a low degreeof selectivity as regards the type of substitution in the anhydroglucoseunit.

[0011] Conventional heterogeneous esterifications are often conducted inwater as a suspension medium (U.S. Pat. No. 5,703,226, 1997).Homogeneous reactions are conducted in organic solvents or in theacylating reagent directly (U.S. Pat. No. 5,714,601, 1998; WO 96/14342).The corresponding carboxylic anhydrides or vinyl esters are generallyused as acylating agents. Esterifications or transesterifications ofthis type are mostly catalysed by alkalies, wherein suitable catalystsinclude alkali hydroxides, salts of mineral acids or organic amines.

[0012] The object of the present invention was to enable esters ofoligo- and polysaccharides to be obtained which are homogeneously andregioselectively substituted at the C2 position of the anhydroglucoseunit (AGU). Success has now been achieved according to the invention inproducing esters of oligo- and polysaccharides which are homogeneouslyand regioselectively substituted at the C2 position of the AGU. Thepresent invention therefore relates to oligo- and polysaccharide esterswhich are esterified regioselectively, with ester groups at the C2position of the AGU preferably amounting to at least 90%, mostpreferably 90 to 98%, of the total degree of substitution (partialaverage degree of substitution AS at the C2 position of the AGU withrespect to the total AS). The oligo- and polysaccharide esters which areregioselectively substituted at the C2 position and which areparticularly preferred according to the invention are esters of starchor starch derivatives, particularly hydroxyethylstarch orhydroxypropylstarch. The present invention also relates to esters, whichare regioselectively substituted at the C2 position, of cellulose,cellulose esters or cellulose ethers, particularly of hydroxyethylcellulose, methyl cellulose, pullulan or maltose. The oligo- andpolysaccharide esters according to the invention are obtainable byreaction with esterification reagents from the group comprising vinylesters, carboxylic anhydrides and trihalogenoethyl esters, as well aslactones, which are described in more detail below in the description ofthe method. Oligo- and polysaccharide esters which regioselectivelysubstituted at the C2 position and which are particularly preferredaccording to the invention are 2-O-propionylstarch, 2-O-butyrylstarch,2-O-benzoylstarch, 2-O-laurylstarch, 2-O-methoxycarbonylstarch,2-O-acryloylstarch and 2-O-methacryloylstarch, most preferably2-O-acetylstarch. According to the invention, the oligo- andpolysaccharides which are regioselectively substituted at the C2position can be esterified at the remaining OH groups of the AGU withother ester groups which are not identical to the ester groups at the C2position.

[0013] The present invention further relates to a method of producingthe compounds according to the invention in the presence of an organicsolvent, which method is catalysed by enzyme or salt and is carried outon a dissolved or highly swollen oligo- or polysaccharide.

[0014] Starting from the disadvantages of the known methods which weredescribed above, e.g. low yields of product and low degrees ofregioselectivity, it has thus proved possible, using catalysis by meansof enzymes, to effect the esterification of oligo- and polysaccharides,which are dissolved in organic solvents or which are strongly swollen,regioselectively at the secondary hydroxyl group of the C2 position, andhas also proved possible to effect said esterification in combinationwith the esterification of the primary hydroxyl group of the C6 positionof the AGU. The method results in very high product yields, and thepartial degrees of substitution can be varied and adjusted over widelimits. Particularly high regioselectivities are achieved with oligo-and polysaccharides which comprise an α-(1,4)-glycoside linkage,particularly starch. However, the method can also be applied to oligo-and polysaccharides which comprise a β-(1,4)-glycoside linkage. Organicsolvents which are suitable in principle are those in which the oligo-and polysaccharides which are used exhibit considerable swelling ordissolve, and in which the enzymes used exhibit satisfactory activity.Polar organic solvents such as Dimethyl sulphoxide (DMSO),N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), pyridine andN-methylmorpholine-N-oxide (NMMO) can therefore be used as solvents, ascan mixtures of the aforementioned solvents. DMSO is preferred. Theenzymes surprisingly exhibit high activity, particularly in DMSO, andproducts which are esterified strictly regioselectively are obtained ina single-stage reaction. These esters can be converted intoregioselectively substituted mixed derivatives, such as esters andethers, by subsequent, selective reactions.

[0015] The esterification can be carried out on polysaccharides,preferably on starch from various natural sources and with wide range ofamylose contents and molecular weights, and on starch derivatives,particularly hydroxyethylstarches or hydroxypropylstarches, and can alsobe carried out on cellulose, cellulose derivatives, pullulan, pullulanderivatives and oligosaccharides.

[0016] Proteases of all types can be used as enzymes. The preferredproteases are serine-, cysteine-, asparagine- and metalloproteases. Theproteases are preferably dissolved in a phosphate buffer, oralternatively in a carbonate buffer, within a pH range of 4-9 dependingon the enzyme, preferably at pH 7-8, and are subsequently lyophilised.The preferred proteases are proteinase N and subtilisin of Bacillussubtilis, proteinase 2A of Aspergillus oryzae, proteinase 6 ofAspergillus sp., a-chymotrypsin, papain, renin and thermolysin.

[0017] Esters of general formula

[0018] are preferably used as esterification reagents, wherein R²preferably denotes a saturated or unsaturated alkyl group comprising 2to 6 C atoms, or an unsaturated or saturated, branched or unbranchedtrihalogenoalkane radical comprising 2 to 4 C atoms, particularly vinyl,trihalogenoethyl or alkyl. R¹ is preferably an alkyl group comprising2-18 C atoms, which can be saturated, unsaturated, straight-chain,branched or cyclic and which can optionally be substituted, or an arylgroup (which is optionally substituted). R¹ is most preferably selectedfrom the group comprising acetyl, propyl, butyryl, vinyl, methacryl,cinnamoyl, pivaloyl and cyclohexyl. When esters of alkenoic acids areused, the double bonds can also be utilised for polymerisation in orderto build up network structures. This option also exists when usingesters of dicarboxylic acids comprising the R² radical, such as vinyladipate, wherein crosslinking can be effected uniformly in the specialpolysaccharide.

[0019] Esterification reagents which are particularly suitable includevinyl acetate, vinyl propionate, vinyl laurate, vinyl butanoate, vinylstearate, vinyl benzoate, vinyl acrylate, vinyl methacrylate, vinylcrotonate, vinyl pivalate and divinyl pivalate.

[0020] Other esterification reagents include carboxylic anhydrides,preferably acetic anhydride, propionic anhydride, succinic anhydride anditaconic anhydride, as well as reactive lactones, preferablypropiolactone and a-angelicalactone. In the case of carboxylicanhydrides, substitution also occurs at the C6 position of the AGU aswell as at the C2 position.

[0021] N-isopropylidene compounds of general formula

[0022] can also be used as esterification reagents, whereupon thecorresponding carboxylic acid esters of the oligo- and polysaccharidesare formed. R¹ is preferably an alkyl group comprising 2-18 C atoms,which can be saturated, unsaturated, straight-chain, branched or cyclicand which is optionally substituted, or can be an aryl group (which isoptionally substituted). R¹ is most preferably selected from the groupcomprising acetyl, propyl, butyryl, vinyl, methacryl, cinnamoyl,pivaloyl and cyclohexyl.

[0023] Particularly suitable esterification reagents from the groupcomprising N-isopropylidene compounds include N-isopropylidene-O-methylcarbonate, N-isopropylidene-O-ethyl carbonate andN-isopropylidene-O-benzyl carbonate.

[0024] The method according to the invention is characterised in thatthe substrate is dissolved in a polar organic solvent—which is DMSO inthe case of starch—to which the enzyme and the transesterificationreagent are added, and is subsequently incubated. The incubationtemperature is between 20° C. and 85° C., and is preferably within therange from 20 to 45° C., particularly in the interval from 35 to 45° C.The times of reaction range from 2 to 100 hours, whereupon a conversionof about 50% is achieved with respect to the acylating reagent.Alternatively, the transesterification reagent can be activated by theenzyme in a preceding step. After the reaction is complete, the enzymeis separated by liquid-solid separation (e.g. centrifugation,filtration). The product is isolated by precipitation and is washed anddried. The solvents which remain can be worked-up by distillation andcan subsequently be recycled to the esterification process. The enzymecan thus be used in a cyclic process without loss of activity.

[0025] When the reaction in the aforementioned system is conducted at atemperature of 40° C., chemical esterification also occurs in additionto enzyme-catalysed esterification. This chemical esterification occurswith the production of degrees of substitution ranging from a fewpercent up to a maximum total degree of substitution corresponding toAS=0.25. This results in the esterification of other hydroxyl groupswhich are present in the molecule. This chemical esterification can besubstantially suppressed if the reaction is conducted at lowertemperatures (20-25° C.), or is preferably suppressed by conducting thereaction in systems which are almost anhydrous (water content <0.01%).

[0026] A partial degree of substitution of up to AS=1.0 at the C2position of the AGU can be achieved in oligo- and polysaccharides by themethod according to the invention. Any desired partial AS≦1.0 at the C2position of the AGU can be achieved via the molar equivalents ofacylating reagent which are used (Table 1). TABLE 1 Molar equivalents ofvinyl acetate and AS_(acetate) values which can be achieved during theproduction of 2-O-acetylstarch with proteinase N in DMSO at 39° C. andat a time of reaction of 70 hours. Molar equivalents vinyl acetate 0.51.0 1.5 2.3 4.0 AS_(acetate) 0.3 0.5 0.7 1.0 1.1

[0027] The partial AS in the C2 position of the AGU can also be adjustedvia the reaction kinetics as well as via the molar equivalents, i.e.desired AS values ≦1.0 can be achieved depending on the time at whichthe reaction is stopped (Table 2). TABLE 2 Time of reaction andAS_(acetate) values which can be achieved during the production of2-O-acetylstarch with proteinase N in DMSO at 39° C. and 2.3 molarequivalents of vinyl acetate. Time of reaction (hours) 2 5 10 20 30 70AS_(acetate) 0.1 0.3 0.5 0.8 0.9 1.0

[0028] Verification of the regioselectivity of the enzymaticallycatalysed esterification reaction was effected on the intact oligo- orpolysaccharide by one- and multi-dimensional NMR spectrometry. For thispurpose, the remaining free hydroxyl groups were esterified with asuitable carboxylic anhydride, for example with propionic anhydride forsaccharide acetates or with acetic anhydride for saccharide benzoates orfor other saccharide acylates. These mixed esters are soluble inchloroform and can be investigated by NMR spectrometry. After evaluatingthe signals of the AGU protons as hydrocarbons via ¹H/¹H and ¹H/¹³Ccorrelation, the corresponding acyl groups can be assigned to theirposition on the AGU with the aid of a ¹H/¹³C multiple bond correlationwhich is detected using ¹H (HMBC technique) (Carbohydr. Res. 224 (1992),277-283).

[0029] In a variant of the method according to the invention, productionis effected catalysed by salts only, without further addition of enzyme.Degress of substitution <1.0 at the C2 position are thereby achieved.

[0030] As distinct from enzyme catalysis, regioselectivity here iscontrolled via the state of dissolution of the oligo- andpolysaccharides in a polar organic solvent, preferably in DMSO.Interactions between the solvents and the AGU components increase theacidity of the proton of the hydroxyl group in the C2 position of theAGU (J. Am. Chem. Soc. 98 (1976), 4386). By employing a suitable salt asa catalyst, complete esterification of this position can then beeffected, wherein it is possible either to employ reaction kineticscontrol or to control the reaction via the type and amount of catalyst(Table 3). The salt is usually present in a concentration of 1-10% byweight, preferably 2-5% by weight, with respect to the startingmaterial. TABLE 3 Dependence of the regioselectivity of acetylation ofstarch (Hylon VII) by vinyl acetate (2.3 molar equivalents) on the timeof reaction and on the type and amount of catalyst Time of Catalystreaction Amount Starch acidity (hours) Type (mol %)¹⁾ AS_(total) ²*AS_(C2) ²** 5 Na₂HPO₄ 10 0.52 0.52 5 Na₂HPO₄ 50 0.92 0.92 5 Na₂HPO₄ 1000.95 0.95 70 Na₂HPO₄ 5 1.00 1.00 4 Na₂CO₃ 10 0.45 0.45 4 Na₂CO₃ 20 0.950.95 4 Na₂CO₃ 50 1.51 0.90. 0.5 K₂CO₃ 10 0.70 0.70 1 K₂CO₃ 10 1.40 0.90

[0031] The method results in higher product yields, and the partialdegree of substitution can be adjusted in a defined manner. Suitablesolvents for carrying out the method include dimethylsulphoxide (DMSO),N,N-dimethylformamide (DMF) and N,N-dimethyl-acetamide (DMA).

[0032] The method can be applied to polysaccharides, preferably tostarches with different amylose contents and molecular weights, tostarch derivatives such as hydroxyethylstarch or hydroxypropylstarch,and to pullulan and pullulan derivatives and oligosaccharides such ascyclodextrin.

[0033] Suitable catalysts include salts of inorganic mineral acids,salts of carboxylic acids and carbonates of the alkali and alkalineearth metals. The preferred salts are Na₂HPO₄, CaHPO₄, Na₂CO₃,MgCO₃(NH₄Cl)₂CO₃, Na₂SO₂, NH₄Cl, NaBr, NaCl and LiCl, as well as sodiumcitrate. In order to suppress esterification at other hydroxyl groups,e.g. in the C₃ or C₆ position, when using salts of weak acids ascatalysts, a maximum of only 10 mol % must be used with respect to theweight of oligo- or polysaccharide to be reacted, and at the same time adefined time of reaction must be adhered to.

[0034] Esters of general formula

[0035] are preferably used as esterification reagents, wherein R² candenote vinyl, trihalogenoethyl or alkyl for example. Examples of R¹include an alkyl group comprising 2-18 C atoms, which can be saturated,unsaturated, straight-chain, branched or cyclic (and which is optionallysubstituted), or an aryl group (which is optionally substituted). Whenesters of alkenoic acids are used, the double bonds can also be utilisedfor polymerisation in order to build up network structures. This optionalso exists when using esters of dicarboxylic acids comprising the R²radical, such as vinyl adipate, wherein crosslinking can be effecteduniformly in the special polysaccharide.

[0036] Other esterification reagents include carboxylic anhydrides, forexample acetic anhydride, propionic anhydride, succinic anhydride anditaconic anhydride, as well as reactive lactones, preferablypropiolactone and α-angelicalactone. In the case of carboxylicanhydrides, substitution also occurs at the C6 position of the AGU aswell as at the C2 position.

[0037] N-isopropylidene compounds of general formula

[0038] can also be used as esterification reagents, wherein thecorresponding carboxylic acid esters of the oligo- and polysaccharidesare formed. Examples of R¹ include alkyl groups comprising 2-18 C atoms,which can be saturated, unsaturated, straight-chain, branched or cyclic(and which are optionally substituted), and aryl groups (which areoptionally substituted).

[0039] The method according to the invention is characterised in thatoligo- or polysaccharides are regioselectively esterified at thehydroxyl group of position C2 of the anhydroglucose unit by activeesters in polar organic solvents—preferably DMSO—using salts ascatalysts. The reaction temperature is between 20° C. and 100° C., andis preferably within the range from 30 to 50° C. The times of reactionrange from 0.5 to 100 hours, depending on the reaction temperature andon the catalyst used. The catalyst is separated by liquid-solidseparation (e.g. centrifugation, filtration). Alternatively, a definedamount of water can also be added to the precipitant, in order todissolve out the catalyst. The resulting ester is isolated byprecipitation and is washed and dried.

[0040] The solvents which remain can be worked-up by distillation andcan subsequently be recycled to the esterification process.

[0041] With the method according to the invention, a partial degree ofsubstitution at the C2 position of the AGU of up to AS=1.0 can beachieved according to choice via the reaction kinetics or via the molarequivalents of transesterification reagent used.

[0042] Regioselectivity was verified by means of two-dimensional NMRspectrometry. For this purpose, the acylated polysaccharide had to becompletely propionylated in the case of acetates, or acetylated in thecase of other polysaccharide esters. In this manner, the substitutionsite can be unambiguously verified by means of multiple bond correlation(Carbohydr. Res. 224 (1992), 277-283).

[0043] The products which are obtained by the method according to theinvention, e.g. starch acetates, can be decomposed by amylases. At asuitable molecular weight of the starch and when substitution iseffected strictly at the C2 position, starch acetates are suitable asblood plasma expanders. 2-O-acetylstarch is particularly suitable forthis application. Therefore, the present invention further relates tothe use of 2-O-acetylstarch as a blood plasma expander. Moreover,biodegradable plastics can be synthesised from starch acylates.Membranes having a substantially uniform structure can be synthesised bycrosslinking processes. Absorbents for different applications can alsobe produced by treatment to form further derivatives. Starch acetateswhich have thermoplastic properties can conceivably be used in thepharmaceutical industry as active ingredient retardants.

[0044] Regioselectively substituted cyclodextrin esters can be used inthe pharmaceutical industry as carriers for pharmaceutical activeingredients. Furthermore, compounds of high molecular weight can besynthesised, in the manner of copolymers, which could be suitable forchromatography (e.g. for the separation of enantiomers).

EXAMPLES Example 1

[0045] 40 g starch (Hylon VII, a native maize starch with a high amylosecontent manufactured by National Starch & Chemical) were heated in 2litres DMSO to 80° C. until a clear solution was formed. After cooling,54 ml vinyl acetate and 750 mg proteinase N of Bacillus subtilis wereadded (the protease was activated by dissolving it in a phosphate bufferpH=7.8; c=0.15 M) and subsequent lyophilisation; the actual amount ofenzyme weighed in was therefore 1.5 g). The mixture was shaken for 70hours at 39° C. After removing the enzyme by centrifugation, the clearcentrifugate was precipitated. The 2-O-acetylstarch was filtered offunder suction, washed, and finally dried under vacuum.

[0046] 46 g 2-O-acetylstarch were obtained which had an AS=1.0.

Example 2

[0047] 40 g starch (Hylon VII) were heated in 2 litres DMSO to 80° C.until a clear solution was formed. 54 ml vinyl acetate and 750 mgproteinase N of Bacillus subtilis were added (see Example 1 for theactivation of the protease). The mixture was shaken for 20 hours at 80°C. After removing the enzyme by centrifugation, the clear centrifugatewas precipitated. The 2-O-acetylstarch was filtered off under suction,washed, and finally dried under vacuum.

[0048] 46 g 2-O-acetylstarch were obtained which had an AS=1.0.

Example 3

[0049] 2 g b-cyclodextrin (manufactured by Fluka) were dissolved in 20ml DMSO, and 2.7 ml vinyl acetate and 37 mg proteinase N of Bacillussubtilis were subsequently added (see Example 1 for the activation ofthe protease). The mixture was shaken for 70 hours at 39° C. Afterremoving the enzyme by centrifugation, the clear centrifugate wasconcentrated, and the product was precipitated, washed, and finallydried under vacuum. 2.1 g heptakis-2-O-acetyl-b-cyclodextrin wereobtained.

Example 4

[0050] 2 g b-cyclodextrin were dissolved in 20 ml DMSO, and 2.7 ml vinylacetate and 37 mg proteinase N of Bacillus subtilis were subsequentlyadded (see Example 1 for the activation of the protease). The mixturewas shaken for 70 hours at 39° C. After removing the enzyme bycentrifugation, the clear centrifugate was concentrated, and the productwas precipitated, washed, and finally dried under vacuum. 2.1 gheptakis-2-O-acetyl-b-cyclodextrin were obtained.

Example 5

[0051] 2 g starch (Hylon VII) were heated in 40 ml DMSO to 80° C. untila clear solution was formed. After cooling, 12.5 g 2,2,2-trichloroethylacetate and 37 mg proteinase N of Bacillus subtilis were added (seeExample 1 for the activation of the protease). The mixture was shakenfor 70 hours at 39° C. After removing the enzyme by centrifugation, theclear centrifugate was precipitated. The 2-O-acetylstarch was filteredoff under suction, washed, and finally dried under vacuum.

[0052] 2.0 g 2-O-acetylstarch were obtained which had an AS=0.4.

Example 6

[0053] 2 g starch (Hylon VII) were heated in 40 ml DMSO to 80° C. untila clear solution was formed. After cooling, 1.9 gN-isopropylidene-O-methyl carbonate and 37 mg proteinase N of Bacillussubtilis were added (see Example 1 for the activation of the protease).The mixture was shaken for 70 hours at 39° C. After removing the enzymeby centrifugation, the clear centrifugate was precipitated. The starchderivative was filtered off under suction, washed, and finally driedunder vacuum.

[0054] 2.0 g 2-O-methoxycarbonylstarch were obtained which had an AS0.4.

Example 7

[0055] 2 g starch (Hylon VII) were heated in 40 ml DMSO to 80° C. untila clear solution was formed. After cooling, 2.7 ml vinyl acetate and 37mg thermolysin were added (see Example 1 for the activation of theprotease). The mixture was shaken for 70 hours at 39° C. After removingthe enzyme by centrifugation, the clear centrifugate was precipitated.The acetylstarch was filtered off under suction, washed, and finallydried under vacuum.

[0056] 2.4 g 2,6-O-diacetylstarch were obtained which had an AS=1.0 atthe C2 position and an AS=0.4 at the C6 position.

Example 8

[0057] 2 g starch (Hylon VII) were heated in 40 ml DMSO to 80° C. untila clear solution was formed. After cooling, 2.7 ml acetic anhydride and37 mg proteinase N of Bacillus subtilis were added (see Example 1 forthe activation of the protease). The mixture was shaken for 70 hours at39° C. After removing the enzyme by centrifugation, the clearcentrifugate was precipitated. The acetylstarch was filtered off undersuction, washed, and finally dried under vacuum.

[0058] 2.0 g acetylstarch were obtained which had an AS=0.7 at the C2and C6 positions of the AGU.

Example 9

[0059] 0.3 g of enzymatically produced starch acetate (Example 1) wassuspended in 5 ml pyridine. 0.1 g dimethylaminopyridine (DMAP) and 5 mlpropionic anhydride were added to this suspension, which was stirred for20 hours at 90° C. The propionylated starch acetate was precipitated inethanol, intensively washed with ethanol and dried under vacuum. Acompletely substituted 2-O-acetyl-3,6-O-dipropionylstarch was obtained.

[0060] The dried product exhibited no OH valency vibrations in the3200-3600 cm⁻¹ IR range and was soluble in chloroform, which resulted inthe following NMR data:

[0061] AGU: d=5.22(H1), 4.72(H2), 5.36(H3), 3.91-3.95(H4,H5), 4.53(H6),4.24(H6′)

[0062] propionyl at position 6: d=1.18(CH₃), 2.45(CH₂)

[0063] propionyl at position 3: d=1.05(CH₃), 2.20(CH₂)

[0064] acetyl at position 2: d=1.98(CH₃)

[0065] (Bruker DRX 400 NMR spectrometer, 323 K)

Example 10

[0066] 2 g cellulose, dissolved in N-methyl-morpholine-N-oxide (MNO),were diluted with DMSO (ratio by volume: VDMSO:VNMMO=1:1). 2.7 ml vinylacetate and 37 mg proteinase N of Bacillus subtilis (see Example 1 forthe activation of the protease) were subsequently added to the cellulosesolution. This mixture was shaken at a temperature of T=80° C. for aperiod of 24 hours. After precipitating the product in hot water, it wasrepeatedly washed with water and finally dried under vacuum. 1.2 gacetyl cellulose were obtained which had an AS of 0.3.

Example 11

[0067] 106 g starch (Hylon VII, a native maize starch with a highamylose content, manufactured by National Starch & Chemical) weredissolved in 1 litre DMSO at 80° C. After cooling to 40° C., 140 mlvinyl acetate and 5 g Na₂HPO₄ were slowly added. The mixture was stirredfor 70 hours and the insoluble Na₂HPO₄ was removed by centrifugation.The product was precipitated in ethanol, filtered under suction, washedand dried under vacuum. 116 g 2-O-acetylstarch was obtained which had anAS=1.0.

Example 12

[0068] 106 g starch (Hylon VII, a native maize starch with a highamylose content, manufactured by National Starch & Chemical) weredissolved in 1 litre DMSO at 80° C. After cooling to 40° C., 63 ml vinylacetate and 5 g Na₂HPO₄ were slowly added. The mixture was stirred for70 hours and the insoluble Na₂HPO₄ was removed by centrifugation. Theproduct was precipitated in ethanol, filtered under suction, washed anddried under vacuum.

[0069] 102 g 2-O-acetylstarch was obtained which had an AS=0.7.

Example 13

[0070] 2 g p-cyclodextrin (manufactured by Fluka) were dissolved in 20ml DMSO, and 2.7 ml vinyl acetate and 20 mg Na₂HPO₄ were subsequentlyadded. The mixture was stirred for 70 hours at 40° C. After removing theinorganic salt by centrifugation, the centrifugate was concentrated andthe product was precipitated in ethanol, washed and dried under vacuum.2.1 g heptakis-2-O-acetylstarch were obtained.

Example 14

[0071] 2 g dextrin 20 (manufactured by Fluka) were dissolved in 40 mlDMSO at 80° C., and 2.7 ml vinyl acetate and 20 mg Na₂HPO₄ weresubsequently added. The mixture was stirred for 70 hours at 40° C. Afterremoving the inorganic salt by centrifugation, the centrifugate wasconcentrated and the product was precipitated in ethanol, washed anddried under vacuum. 2 g 2-O-acetyldextrin were obtained, which had anAS=1.0.

Example 15

[0072] 2 g starch (Hylon VII, a native maize starch with a high amylosecontent, manufactured by National Starch & Chemical) were dissolved in40 ml DMSO at 80° C. After cooling to 40° C., 2.7 ml vinyl acetate and20 mg NaCl were slowly added. The mixture was stirred for 70 hours andthe insoluble NaCl was removed by centrifugation. The product wasprecipitated in ethanol, filtered under suction, washed and dried undervacuum. 2.1 g 2-O-acetylstarch was obtained which had an AS=1.0.

Example 16

[0073] 2 g starch (Hylon VII, a native maize starch with a high amylosecontent, manufactured by National Starch & Chemical) were dissolved in40 ml DMSO at 80° C. After cooling to 40° C., 2.7 ml vinyl acetate and20 mg Na₂CO₃ were slowly added. The mixture was stirred for 70 hours andthe insoluble Na₂CO₃ was removed by centrifugation. The product wasprecipitated in ethanol, filtered under suction, washed and dried undervacuum. 2.1 g 2-O-acetylstarch was obtained which had an AS=1.0.

1. A regioselectively substituted member selected from the groupconsisting of oligo-saccharide ester and polysaccharide ester, having apartial average degree of substitution AS at the C2 position of itsanhydroglucose unit of at least 90% relative to the total AS.
 2. Theregioselectively substituted member of claim 1 comprising at least onespecie selected from the group consisting of starch and starchderivative.
 3. The regioselectively substituted member of claim 2wherein specie is selected from the group consisting of2-O-propionylstarch, 2-O-butyrylstarch, 2-O-benzoylstarch,2-O-laurylstarch, 2-O-methoxycarbonylstarch, 2-O-acrylolylstarch,2-O-methacrylolylstarch and 2-O-acetylstarch.
 4. The regioselectivelysubstituted member of claim 1 comprising at least one specie selectedfrom the group consisting of cellulose and cellulose derivative.
 5. Amethod of producing the regioselectively substituted member of claim 1comprising reacting in the presence of an enzyme catalyst a dissolved ora highly swollen specie selected from the group consisting ofoligo-saccharide and polysaccharide with an esterification reagent. 6.The method of claim 5 wherein specie is at least one member selectedfrom the group consisting of natural starch, starch derivative,cellulose, cellulose derivative, pullulan and pullulan derivative. 7.The method of claim 5 wherein enzyme is protease.
 8. The method of claim7 wherein protease is selected from the group consisting of proteinaseN, subtilisin, protease 2A, protease 6, a-chymotrypsin, renin, papain,thermolysin and esterase.
 9. The method of claim 5 characterized in thatit is carried out in at least one organic solvent selected from thegroup consisting of dimethylsulphoxide (DMSO), N,N-dimethylformamide(DMF), N,N-dimethylacetamide (DMA), pyridine and N-morpholine-N-oxide(NMMO).
 10. The method of claim 5 further comprising isolating theenzyme upon completion of the reaction.
 11. The method of claim 5wherein esterification reagent is vinyl ester.
 12. The method of claim11 wherein vinyl ester is selected from the group consisting of vinylacetate, vinyl propionate, vinyl laurate, vinyl butanoate, vinylstearate, vinyl benzoate, vinyl acrylate, vinyl methacrylate, vinylcrotonate, vinyl pivalate and divinyl adipate.
 13. The method of claim 5wherein esterification reagent is carboxylic anhydride.
 14. The methodof claim 13 wherein carboxylic anhydride is at least one member selectedfrom the group consisting of acetic anhydride, propionic anhydride,succinic anhydride, itaconic anhydride.
 15. The method of claim 5wherein esterification reagent is at least one member selected from thegroup consisting of trihalogenoethyl esters and lactones.
 16. The methodof claim 15 wherein lactone is at least one member selected from thegroup consisting of propiolactone, butyrolactone, and a-angelicalactone.17. The method of claim 5 wherein esterification reagent is at least onemember selected from the group consisting of O-substitutedN-isopropylidene carbonates, N-isopropylidene-O-methyl carbonate,N-isopropylidene-O-ethyl carbonate and N-isopropylidene-O-benzylcarbonate.
 18. A method of producing a member selected from a firstgroup consisting of oligo-saccharide ester and a polysaccharide esters,which member is regioselectively substituted at the C2 position and hasa degree of substitution lower than 1.0, comprising reacting (i) adissolved or highly swollen saccharide selected from a second groupconsisting of oligo-saccharide and polysaccharide, in the presence ofsalt as catalyst and in an organic solvent with (ii) and esterificationreagent.
 19. The method of claim 18 wherein salt is selected from thegroup consisting of Na₂HPO₄, Na₂CO₃, MgCO₃, (NH₄)₂CO₃, NH₄Cl, NaCl,NaBr, LiCl and sodium citrate.
 20. A blood plasma expander comprisingthe regioselectively substituted ester of claim 1.